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Genes to Cells (2006) 11, 499-512. doi:10.1111/j.1365-2443.2006.00956.x
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Antagonistic regulation of the Drosophila PCNA gene promoter by DREF and Cut

Hirokazu Seto1,2, Yuko Hayashi3, Eunjeong Kwon3,a, Osamu Taguchi4 and Masamitsu Yamaguchi1,2,*

1 Department of Applied Biology, Faculty of Textile Science, and
2 Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
3 Division of Biochemistry, and
4 Division of Molecular Pathology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464-8681, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The gene promoter of Drosophila proliferating cell nuclear antigen (dPCNA) contains several transcriptional regulatory elements, such as upstream regulatory element (URE), DNA replication-related element (DRE, 5'-TATCGATA), and E2F recognition sites. In the present study, a yeast one-hybrid screen using three tandem repeats of DRE in dPCNA promoter as the bait allowed isolation of a cDNA encoding Cut, a Drosophila homolog of mammalian CCAAT-displacement protein (CDP)/Cux. Electrophoretic mobility shift assays showed that Cut bound to both DRE and the sequence 5'-AATCAAAC in URE, with much higher affinity to the former. Measurement of dPCNA promoter activity by transient luciferase expression assays in Drosophila S2 cells after an RNA interference for Cut or DREF showed DREF activates the dPCNA promoter while Cut functions as a repressor. Chromatin immunoprecipitation assays in the presence or absence of 20-hydroxyecdysone further showed both DREF and Cut proteins to be localized in the genomic region containing the dPCNA promoter in S2 cells, especially in the Cut case upon induction of differentiation. These results indicate that Cut functions as a transcriptional repressor of dPCNA gene by binding to the promoter region in the differentiated state, while DREF binds to DRE to promote expression of dPCNA during cell proliferation.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The proliferating cell nuclear antigen (PCNA), an accessory protein of DNA polymerase {delta} is required for replication of simian virus 40 as well as cellular DNA (Jaskulski et al. 1988; Tsurimoto et al. 1990). To date, many studies have provided evidence for an essential function of PCNA in normal DNA replication as a sliding clamp at DNA replication forks (Kelman & O'Donnell 1995), DNA repair (Shivji et al. 1992) and cell cycle regulation (Flores-Rozas et al. 1994; Matsuoka et al. 1994; Waga et al. 1994) by interacting with various enzymes and regulatory proteins (Kelman & Hurwitz 1998; Warbrick 1998). Interaction of PCNA with the chromatin assembly factor 1 (CAF-1) suggests another role in marking of DNA for chromatin assembly during the S phase of the cell cycle (Henderson et al. 1994; Shibahara & Stillman 1999; Zhang et al. 2000).

Previous studies on the Drosophila PCNA (dPCNA) gene have revealed that its promoter region contains multiple regulatory elements, such as the upstream regulatory element (URE), the DNA replication-related element (DRE) (Hirose et al. 1993; Yamaguchi et al. 1995b; Takahashi et al. 1996), E2F-recognition sites (Yamaguchi et al. 1995a), common regulatory factor for DNA replication and DREF (DRE-binding factor) genes (CFDD)-recognition sites (Hayashi et al. 1997), and most recently a Drosophila regulatory factor X2 (dRFX2)-recognition site (Otsuki et al. 2004). It has been demonstrated that the transcription factor Grainyhead (GRH/NTF-1) binds to URE (Bray et al. 1989; Dynlacht et al. 1989; Hayashi et al. 1999). One of the three CFDD sites (sites 1, 2 and 3) in the dPCNA gene promoter, the site 1, was demonstrated to play an important role in promoter activity in both cultured cells and living flies. In addition, CFDD sites are also present in promoters of the DNA polymerase {alpha} and DREF genes, although specific CFDD binding factors have yet to be identified (Hayashi et al. 1997). URE and the E2F sites, CFDD sites, dRFX2 site and DRE appear to be essential for activation of the dPCNA gene promoter in larvae (Yamaguchi et al. 1996). The dRFX2 site is located between URE and DRE in the dPCNA gene promoter and the dRFX2 binding factor contains a characteristic motif which is conserved among regulatory factor X (RFX) family proteins (Reith et al. 1990; Wu & McLeod 1995; Emery et al. 1996; Huang et al. 1998; Durand et al. 2000), known to be present in yeast, fungi, nematodes, mice, humans and Drosophila. The human RFX-binding site has been suggested to be involved in regulation of the human PCNA gene (Labrie et al. 1995; Lee et al. 1998; Liu et al. 1999). Therefore, the function of some RFX family proteins might be conserved during evolution. Over-expression of the DNA-binding domain of dRFX2 in imaginal discs inhibits DNA synthesis but exerts no effects on photoreceptor cell differentiation (Otsuki et al. 2004). These results indicate that dRFX2 plays a role in the G1–S transition and/or in S-phase progression.

DRE appears to be an important regulatory element not only for DNA replication-related genes but also for various other examples involved in the cell cycle (Ohno et al. 1996) and proliferation (Matsukage et al. 1995; Ryu et al. 1997). DRE is essential for dPCNA gene promoter activity throughout development, except for maternal expression in ovary. In addition to DREF, it is reported that boundary element-associated factor of 32 kDa (BEAF32) (Liu et al. 1989; Hirose et al. 1993; Hart et al. 1999) can bind to DRE, suggesting the possibility that there are other factors that could bind to DRE.

In the present study, a yeast one-hybrid screen, using DRE as a bait, allowed isolation of cDNAs encoding not only DREF but also Cut, a Drosophila homolog of mammalian CCAAT-Displacement Protein (CDP)/Cux. Cut, an evolutionarily conserved protein, has been detected in a variety of sites, including external sensory organs, Malpighian tubules, and ovary follicle cells (Blochlinger et al. 1993). Genetic studies in Drosophila melanogaster suggested that it functions to determine cell type specification in these tissues (Bodmer et al. 1987; Blochlinger et al. 1991; Liu et al. 1991; Ludlow et al. 1996; Nepveu 2001). CDP, a highly conserved homeodomain protein of cut homolog in mammals, was also found to function as a transcriptional repressor by recruitment of a histone deacetylase activity (Nishio & Walsh 2004). Our present electrophoretic mobility shift assays (EMSA) revealed that Cut binds to both DRE and the sequence 5'-AATCAAAC in URE of the dPCNA gene promoter and transient luciferase expression assays using constructs containing promoter regions of dPCNA gene after knocking down either DREF or cut by RNA interference in Drosophila S2 cells demonstrated that DREF functions as a positive regulator while Cut acts as a negative regulator of the dPCNA promoter. In addition, Chromatin immunoprecipitation (ChIP) assays suggested that Cut and DREF bind differentially to the promoter region depending on exposure to 20-hydroxyecdysone, a molting hormone causing differentiation.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Isolation of cDNAs encoding binding factors to DRE in the dPCNA promoter

We carried out yeast one-hybrid screens to isolate cDNAs encoding the protein responsible for the DRE binding. We screened a Drosophila third instar larval cDNA library using three tandem copies of DRE as the target binding sequence. With a total of 7.95 x 106 independent clones, four positive examples were identified, all of which were partially sequenced. Three positive clones were found to encode DREF, while one clone encoded the cut gene product (Blochlinger et al. 1988) (GenBankTM GeneID: NM 080025, FlyBase ID: FBgn0004198). No cDNA encoding BEAF32 was isolated by this screening. The isolated clone designated as pACT-Cut1057–2175 was transformed into yeast strains containing the 5UREL-reporter, the 3URE-reporter, 3DRE-reporter, three copies of the CFDD-1 (3CFDD)-reporter, or four copies of the E2F-P (4E2F)-reporter. Only with the 3DRE-reporter did the transformants grow in the presence of 40 mM 3-aminotriazole (Fig. 1A), which suggests that Cut specifically binds to DRE in yeast cells. Due to stringent conditions by relatively high concentration of 3-aminotriazole, it might not have allowed us to detect interaction between Cut and URE-reporter, although a weak interaction between them is detectable in EMSA as described below.


Figure 1
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  		Figure 1  One-hybrid assay and Complex formation: Cut-dPCNA (–149/–62) and DREF-dPCNA (–149/–62). (A) PACT-CUT1057–2175 plasmid DNA was transformed into yeast strains containing the 5UREL-His, the 3URE-His, 3DRE-His, three copies of the oligonucleotide CFDD-1 (3CFDD)-His, or four copies of the oligonucleotide E2F-P (4E2F)-His as indicated on the left. Each transformant was seeded on a synthetic dropout agar plate without histidine but containing 0 or 40 mM 3-aminotriazole (3-AT) and incubated for 3 days at 30 °C. (B) 32P-labeled double stranded oligonucleotides carrying the –149/–62 dPCNA region were incubated with Kc cell nuclear extracts (4 µg of protein) after incubation with the indicated amounts of mouse anti-Cut IgG (lanes e–g) or culture supernatants of hybridoma cells producing anti-DREF IgG (lanes j–l) or control mouse IgG (lanes b–d). r.p.m.-I: control for culture supernatant, anti-DREF antibody. 0 or (–) minus are no antibody controls.

 
Cut and DREF binds to dPCNA gene promoter region containing DRE

To detect factors binding to dPCNA gene promoter regions, an oligonucleotide probe between –149 and –62 was amplified by PCR and used for EMSA. Two shifted bands were detected with the oligonucleotide and embryo nuclear extracts (Fig. 1B, lane a). To obtain direct evidence for binding of Cut and DREF to the dPCNA gene promoter region, we then conducted EMSA with anti-Cut or anti-DREF antibodies (Fig. 1B). On addition of the anti-Cut antibody to the binding reaction, the slower migrating band was diminished (Fig. 1B, lanes e–g), while addition of the anti-DREF antibody reduced the faster migrating band (Fig. 1B, lanes j–l). However, the addition of control IgG exerted no effect on complex formation (Fig. 1B, lanes b–d). Thus both Cut and DREF can bind to the region from –149 to –62 of the dPCNA gene promoter.

The oligonucleotide used for EMSA contains several transcriptional regulatory elements of the dPCNA gene such as URE (–168 to –118), DRE (–100 to –93) and three CFDD sites (–134 to –127, –100 to –92, –84 to –77) (Hayashi et al. 1997). To establish the binding specificity of Cut or DREF to the dPCNA gene promoter region, we examined EMSA with non-radio labeled competitor oligonucleotides, DRE-P, CFDD-1 or URE (Fig. 2A,B). The results showed that DRE-P diminished bands corresponding to both Cut-oligonucleotide and DREF-oligonucleotide complexes (Fig. 2C, lanes e–g) whereas the CFDD-1 oligonucleotide exerted no effect (Fig. 2C, lanes h–j). Interestingly, URE oligonucleotide could diminish a band corresponding to Cut-oligonucleotide complex dose dependently, but not as strongly as DRE-P, while exerting no effect on formation of DREF-oligonucleotide complexes (Fig. 2C, lanes k–m). These results indicate that both DREF and Cut bind DRE in the dPCNA gene promoter. Although Cut can also bind to URE, its affinity is much weaker than that for DRE.


Figure 2
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  		Figure 2  Differences in binding sites between Cut and DREF on the dPCNA promoter. (A) The diagram shows the structure of the dPCNA gene promoter region. The vertical line with a horizontal arrow indicates the transcription initiation site. The open box indicates the dPCNA gene. CFDD sites 1, 2, and 3 are indicated with black boxes. Two E2F-recognition sites, DRE and URE, are also shown. (B) Nucleotide sequences in and around the CFDD 1, DRE and URE in dPCNA gene. Numbers with vertical lines indicate locations relative to the transcription initiation site. Boxes indicate nucleotide sequences of CFDD sites 1, 2, and 3. (C) Radiolabeled double stranded oligonucleotides carrying the wild-type –149/–62 site were incubated with Kc cell nuclear extracts (4 µg of protein) in the presence or absence (0) of the indicated amount of competitor oligonucleotides (see the top of each lane). Complexes between Cut and the oligonucleotide –149/–62 (Cut) and between DREF and the oligonucleotide –149/–62 (DREF) are shown by arrows.

 
Sequence specificity of Cut-DNA binding

To examine effects of mutations in DRE, a base substitution mutation, DRE-PM and internal deletion mutants, mut{Delta}1(–96) and mut{Delta}1(–98) were introduced and used as competitors (Fig. 3). The slower migrating bands by Cut protein were not diminished by the addition of oligonucleotides carrying any of the mutations, while the faster migrating bands by DREF protein were diminished by DRE-PM or mut{Delta}1(–98) dose dependently, but not by mut{Delta}1(–96) under these conditions (Fig. 3B). Both DRE-P oligonucleotide and triple tandem repeats of DRE (3DRE-P) strongly competed against complex formation for both proteins. These results indicate that all eight nucleotides in the sequence (5'-TATCGATA) are strictly required for Cut-binding, while the sequence requirement for DREF-binding appears to be rather flexible.


Figure 3
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  		Figure 3  Effects of mutations in the DRE site on complex formation with –149/–62 oligonucleotides. (A) Nucleotide sequences in and around the DRE of the wild-type and mutant oligonucleotides are shown. Nucleotides with substitution for the wild-type sequence are indicated in lower case letters. Deletion points in eight base pairs for DRE are indicated by hyphens. Nucleotide sequences of DRE are indicated by the box. Three tandem repeats of the sequence containing DRE site are constructed. DRE as well as its base-substituted derivatives are underlined. (B) Radio-labeled double stranded oligonucleotides carrying the wild-type –149/–62 region were incubated with Kc cell nuclear extracts (4 µg of protein) in the presence or absence (0) of the indicated amount of competitor oligonucleotides (see the top of each lane).

 
Since Cut has affinity for URE as described above, base substitution mutations (mut{alpha}, ß, {gamma}, {delta}, and {varepsilon}) were introduced in and around URE and used as competitors (Fig. 4). It is known that the URE site of dPCNA gene promoter contains the CAATCA sequence (–135 to –130) and CFDD site 3 (–134 to –127) (Hayashi et al. 1997). Neither mut{gamma} nor mut{delta} diminished the slower migrating band representing the Cut-oligonucleotide complex, while mut{alpha}, ß and {varepsilon} diminished the band dose dependently (Fig. 4B). The results indicate that the nucleotide sequence 5'-AATCAAAC (–134 to –127) is required for Cut-binding. This sequence perfectly matches the CFDD site 3.


Figure 4
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  		Figure 4  Effects of mutations in URE on complex formation with –149/–62 oligonucleotides. (A) Nucleotide sequences in and around URE of wild-type and mutant oligonucleotides are shown. Nucleotides substituted for the wild-type sequence are indicated in lower case letters. The Cut binding site (5'-CAATCA) sequence is indicated. (B) Radio-labeled double stranded oligonucleotides carrying the wild-type –149/–62 region were incubated with Kc cell nuclear extracts (4 µg of protein) in the presence or absence (0) of the indicated amount of competitor oligonucleotides (see the top of each lane).

 
Effects of knock down of Cut or DREF genes on dPCNA gene promoter activity in S2 cells

Efficiency of knock down of cut and DREF genes was monitored by measuring levels of Cut and DREF proteins in Drosophila S2 cells. Western immunoblot analyses with anti-Cut and anti-DREF antibodies were conducted after treating cells with CutdsRNA, DREFdsRNA, LacZdsRNA (control) or no dsRNA (mock control). The results showed both Cut and DREF dsRNAs to significantly reduce the expression level of Cut and DREF proteins, respectively, while LacZdsRNA or mock treatment exerted no effects (Fig. 5).


Figure 5
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  		Figure 5  Western immunoblot analysis of cells treated with CutdsRNA and DREFdsRNA. Fifteen micrograms each of dsRNAs, CutdsRNA, DREFdsRNA, LacZdsRNA, were transfected into S2 cells. As a control, no dsRNA treatment (Mock) was performed in parallel. Two, three and four days after dsRNA treatment, S2 cells were harvested and subjected to SDS-PAGE and proteins were probed with anti-Cut, anti-DREF, and anti-tubulin antibodies.

 
To evaluate the function of Cut and DREF in dPCNA gene promoter activity, transient luciferase expression assays were conducted with plasmids carrying the dPCNA gene promoter or its 5' deletion derivatives fused with the luciferase reporter gene, after treating Drosophila S2 cells with CutdsRNA (Fig. 6A), DREFdsRNA (Fig. 6B), LacZdsRNA (control) (Fig. 6C) or no dsRNA (mock) (Fig. 6D). Treatment of cells with DREFdsRNA reduced dPCNA promoter activity with –119PCNA-luc to 15% (Fig. 6B), indicating that DREF is required for dPCNA promoter activity. Although deletion up to –116 exerted no significant effect on the extent of reduction of promoter activity with DREFdsRNA, when we used –70PCNA-luc as a reporter, reduction of promoter activity was no longer observed, indicating that DRE is required for the effect of DREFdsRNA. In contrast, treatment of cells with CutdsRNA increased dPCNA promoter activity by 1.5-(–168PCNA-luc) to three fold (–70PCNA-luc) (Fig. 6A), while LacZdsRNA or mock treatment exerted no effect on dPCNA promoter activity (Fig. 6C,D). Furthermore, the level of endogenous PCNA protein was increased by treatment of cells with CutdsRNA, but reduced with DREFdsRNA (Fig. 6E). These results indicate that DREF functions as an activator for the dPCNA gene, while Cut functions as a negative regulator for the dPCNA gene.


Figure 6
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  		Figure 6  Effects of knock down of DREF and cut on dPCNA gene promoter activity. Drosophila S2 cells were treated with 3 µg each of (A) CutdsRNA, (B) DREFdsRNA or (C) LacZdsRNA prior to transfection of the indicated reporter plasmids harboring various regions of the dPCNA promoter. At 48 h after the transfection, the cells were harvested and subjected to luciferase assays. (D) Mock indicates no treatment of cells with double stranded RNA. Luciferase activity was normalized to Renilla luciferase activity and expressed as activity relative to that with mock alone scored as 100. Averaged values obtained from nine independent wells with standard deviations are shown by closed bars with vertical lines. (E) Immunoprecipitates with anti-Cut or anti-DREF antibody from cells treated with either CutDsRNA or DREFdsRNA were subjected to Western immunoblot analysis with the anti-PCNA antibody.

 
Cut and DREF protein loading on the genomic region containing the dPCNA promoter with and without ecdysone treatment of S2 Cells

It is well known that 20-hydroxyecdysone, an insect molting hormone, can induce differentiation of cultured Drosophila S2 cells (Berger et al. 1978), providing a cellular model system to study mechanisms involved in the switch from proliferation to differentiation. Interruption of S2 cell proliferation by the addition of 20-hydroxyecdysone is accompanied by an arrest of DNA synthesis, deduced from the measurement of tritium labeled thymidine incorporation performed previously (Gvozdev et al. 1974; Rosset 1978; Munsch & Cade-Treyer 1982). To test the effect of ecdysone hormone on dPCNA promoter activity in S2 cells, we conducted transient luciferase expression assays with –168PCNA-luc plasmid in the presence or absence of 20-hydroxyecdysone. As expected, the addition of 2 µM of 20-hydroxyecdysone reduced the luciferase activity to 30% (Fig. 7A) and this effect was observed up to 10 µM.


Figure 7
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  		Figure 7  Localization of Cut and DREF proteins in the genomic region containing the dPCNA promoter in S2 cells in the presence or absence of ecdysteroid hormone. (A) Transient luciferase expression assays were conducted to monitor the effects of ecdysteroid hormone on dPCNA promoter activity. Prior to transfecting 50 ng of plasmid –169 dPCNA-luc for each well, S2 cells were cultured with or without 20-hydroxyecdysone. Luciferase activity was normalized to Renilla luciferase activity and expressed as relative to that with 0 µM of 20-hydroxyecdysone. Averaged values obtained from nine independent wells with standard deviations are shown by closed circles with vertical lines. (B) Proliferating S2 cells were harvested after formaldehyde treatment to fix proteins on genomic DNA. ChIPs were performed with anti-DREF antibody ({alpha}DREF) or control IgG (IgG) or without serum. Immunoprecipitates were analyzed for the presence of dPCNA promoter by PCR amplification. (C) Twenty-four hours before formaldehyde fixation, S2 cells were cultured with or without 10 µM of 20-hydroxyecdysone. ChIPs were performed with anti-Cut ({alpha}Cut) or anti-DREF ({alpha}DREF) antibodies or without antibodies. Immunoprecipitates were analyzed for the presence of dPCNA promoter by PCR amplification. A primer pair of rp49 gene was used as a negative control for PCR amplification.

 
As described above, DREF functions as an activator for the dPCNA gene promoter, while Cut functions as a negative regulator. Therefore, Cut may be loaded on to the dPCNA gene promoter region after treatment with 20-hydroxyecdysone. To address this possibility, we conducted ChIP assays with anti-Cut or anti-DREF monoclonal antibodies. Firstly, we established ChIP assay conditions in which the dPCNA promoter region is specifically amplified from the immunoprecipitates with anti-DREF antibody, but not with non-immune control IgG (Fig. 7B). The data also provide the first direct evidence indicating that DREF binds to the dPCNA promoter region in vivo, although this has also been suggested from a number of in vitro binding studies (Hirose et al. 1993; Matsukage et al. 1995; Yamaguchi et al. 1996; Takahashi et al. 1997). Potential binding of Cut to the dPCNA promoter region in vivo is also suggested by the yeast one-hybrid assay and EMSA results described above. Then, we carried out ChIP assays with anti-DREF or anti-Cut antibodies in cells with or without ecdysone. In the latter case, strong signals with the anti-DREF antibody were observed, while only a weak signal was noted with the anti-Cut antibody (Fig. 7C, –ecdysone). In contrast, much stronger signals were obtained in samples with the anti-Cut antibody than with the anti-DREF antibody by adding 20-hydroxyecdysone (Fig. 7C, +ecdysone). Since no significant signals for amplification of rp49 gene in the PCR with any of immunoprecipitates were observed, while inputs revealed a strong signal, we concluded that signals amplified by PCR with dPCNA promoter region revealed specific localization of Cut or DREF on the region. These results taken together suggest that Cut is recruited to the dPCNA gene promoter locus to inhibit its activity during differentiation.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In the study presented here, we identified a Drosophila Cut protein that binds to the multiple dPCNA promoter regions. Thus yeast one-hybrid screening using three tandem repeats of DRE in the dPCNA promoter as a bait allowed isolation of cDNAs encoding both DREF and Cut proteins. Cut, a Drosophila homolog of mammalian CCAAT-displacement protein (CDP)/Cux is known as an evolutionally conserved homeodomain protein which contains unique structural properties, three regions, called Cut repeats (CR1, CR2, and CR3), and the Cut homeodomain (HD) (Blochlinger et al. 1988; Neufeld et al. 1992; Andres et al. 1994; Nepveu 2001). Multiple reports suggest that mammalian CDP/Cut can bind to a wide range of DNA sequences, such as 5'-CCAAT, 5'-ATCGAT, Sp1 sites, and AT rich matrix attachment regions (Dickinson et al. 1997; Liu et al. 1997; Chattopadhyay et al. 1998; Wang et al. 1999). From studies using purified CDP/Cut protein and panels of fusion proteins, it appears that CR1 and CR2 are responsible for binding to the 5'-CCAAT sequence, with 5'-C(A/G)AT site near the 5'-CCAAT binding site required for CCAAT displacement activity (Moon et al. 2000). Our EMSA studies with anti-Cut and anti-DREF antibodies confirmed that the factors binding to the dPCNA promoter region between –149 and –62 are Cut and DREF proteins. This region contains two unique nucleotide sequences, 5'-CAATCA (–135 to –130) and 5'-TATCGATA (–100 to –93). EMSA with non-radioisotope labeled competitor oligomers, DRE-P, CFDD-1, and URE, suggested that Cut binds to both DRE and URE of the dPCNA gene promoter, with much higher affinity to DRE, while DREF binds specifically to DRE but not to URE (Fig. 2C). The sequence specificity of Cut- and DREF-binding determined by EMSA with a set of base substitution mutations and deletion mutants in and around DRE or URE as competitors can be summarized as follows: Firstly, all eight nucleotides of the sequence (5'-TATCGATA) are strictly required for Cut-binding to DRE, while the sequence requirement for DREF-binding to DRE appears to be more flexible. It should be noted that the DRE sequence (5'-TATCGATA) contains 5'-ATCGAT which is reported to have a strong affinity for a mammalian Cut homolog (Harada et al. 1995). Secondly, the nucleotide sequence 5'-AATCAAAC (–134 to –127) in URE is required for Cut-binding, while DREF has no affinity for this sequence. It should also be noted that the determined sequence spans the sequence 5'-CAATCAAAC in URE and it nearly matches the 5'-CCAAT, again with strong affinity for the mammalian Cut homolog (Andres et al. 1994). These findings suggest that Drosophila Cut has similar sequence specificity to that of its mammalian counterpart. In addition, since we still observed CutRNAi effects with deletion of the dPCNA promoter up to –70, the region from –70 to +23 may have another binding sequence to Cut, although further analysis is necessary to clarify this point.

In addition to DREF and Cut, three other protein factors are reported to be involved in regulation of the dPCNA gene promoter. The 32 kDa boundary element associate factor (BEAF 32) can bind to the sequence 5'-ATCGAT in DRE (5'-TATCGATA) and appears to antagonize DREF both in vitro and in vivo (Hart et al. 1999). Grainyhead (GRH) has been identified as a positive transcriptional regulator for the dPCNA gene which binds to the sequence 5'-AAACCAGTTGGCA (–130 to –118) in URE (Hayashi et al. 1999), which at least partially overlaps with the Cut-binding sequence 5'-AATCAAAC (–134 to –127). Armadillo/pangolin is another transcription factor that could positively regulate the dPCNA gene (Kwon et al. 2004). Armadillo/pangolin can bind to the sequence 5'-TCGATAGATCA (–98 to –87), which also partially overlaps with the Cut- and DREF-binding sequence 5'-TATCGATA (–100 to –93), DRE. The contribution of each transcription factor for the dPCNA gene promoter activity could vary with different cell types, possibly depending on the relative abundance of each factor in each cell type.

The data in the present study indicate that DREF functions as an activator for the dPCNA gene, while Cut functions as a negative regulator. Furthermore, we demonstrated by ChIP assay that Cut is recruited to the dPCNA gene promoter locus after induction of differentiation by ecdysone treatment of cells, while DREF mainly localizes to the dPCNA gene promoter in the growing cells. In general, mammalian CDP/Cut proteins have been found to function as transcriptional repressors by competing with activator(s) to occupy binding sites (Mailly et al. 1996). It is proposed that the evolutionarily conserved domains, three cut repeats and a homeodomain, function as specific DNA binding domains (Aufiero et al. 1994; Moon et al. 2000). Mammalian CDP/Cut proteins recruit histone deacetylase (HDAC) activity to promoter regions (Blochlinger et al. 1993) and recently it was reported that CDP/Cut interacts with a histone lysine methyltransferase, G9a (Nishio & Walsh 2004). These known aspects of mammalian CDP/Cut are consistent with our conclusion that Cut functions as a repressor of the dPCNA gene by binding to its promoter region during cell differentiation, while DREF binds to DRE to promote expression of the dPCNA gene for cell proliferation. Here we can propose a model that dPCNA gene expression is regulated by antagonistic mechanisms between DREF and Cut via URE and DRE in the dPCNA promoter region. During cell proliferation, DREF localizes to DRE, and the state of dPCNA gene expression is "on." On the other hand, when cells are in a differentiated state, Cut is recruited to the dPCNA promoter regions, via DRE as well as URE or via around these sites, and acts with other factors, such as HDAC and/or G9a, to switch off dPCNA gene expression (Fig. 8).


Figure 8
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  		Figure 8  Antagonistic regulation of dPCNA gene expression by DREF and Cut. (A) In the proliferating state, DREF binds to DRE in dPCNA gene promoter and recruits other factors that initiate transcription of the gene. Then the expression of the dPCNA gene is "ON." (B) In the differentiated state, Cut localizes to dPCNA gene promoter via URE and DRE or via around these sites. Cut may recruit other factors, such as G9a and HDAC, then the dPCNA promoter is repressed and becomes "OFF."

 
Coordinated shutdown of many cell proliferation-related genes is essential for cellular shift from a proliferating to a resting state. Differentiation signals must control this process and a key transcription factor for proliferation-related genes such as DREF is a likely target for repression. Previously, we reported that the dPCNA gene is repressed by Zerknüllt, a homeodomain protein responsible for differentiation of dorsal tissues during embryogenesis (Yamaguchi et al. 1991). Subsequently, it was reported that Zen down-regulates DREF gene promoter activity to repress dPCNA gene expression (Hirose et al. 1994). More recently we found that a homeodomain protein Distal-less can negatively regulate DREF activity by directly associating with its DNA-binding domain (submitted for publication). In the present study, we found that another homeodomain protein Cut can antagonize function of DREF to inhibit dPCNA gene expression. Since proliferation-related genes carrying DRE are also potential targets of Cut, the antagonistic mechanism might be applicable to many other proliferation-related genes. The repression of proliferation-related genes by homeodomain proteins may explain the differentiation-coupled repression of cell proliferation during development, although the mechanisms for the repression are different in each case.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell culture

Drosophila Kc cells and Schneider (S2) cells were cultured in M3(BF) medium (Cross & Sang 1978), supplemented with 2% and 10% fetal bovine serum, respectively.

Oligonucleotides

(i) The following primer pairs were used for the polymerase chain reaction (PCR) to amplify DNA templates for synthesizing dsRNAs targeting Drosophila cut (750 bp from the initiation codon).

To generate forward direction RNA, the following primer set was used:

5'-TCCCCCGGGGGAGTTGCCAGTTGCAATCGGTGTT (smaI-Cut-FWD-3)

5'-CGGGGTACCCCGATGCAGCCAACATTGCCACAAG (kpnI-Cut-FWD-5)

To generate reverse direction RNA, the following primer set was used:

5'-TCCCCCGGGGGAATGCAGCCAACATTGCCACAAG (smaI-Cut-REV-3)

5'-CGGGGTACCCCGGTTGCCAGTTGCAATCGGTGTT (kpnI-Cut-REV-5)

(ii) To generate a probe that was used to identify sequence specific binding factors in EMSA, the following primer pairs were synthesized to amplify sequence between –149 to –62 of dPCNA promoter region.

The sequence of double-stranded oligonucleotides containing DRE (DRE-P) in the PCNA gene was as described earlier (Yamaguchi et al. 1995a; Yamaguchi et al. 1995b). The oligonucleotide DRE-PM is a two-base substitution derivative of DRE-P (Yamaguchi et al. 1995b). The sequences of double-stranded oligonucleotides containing CFDD site 1 (–87 to –62) in the PCNA gene (Hayashi et al. 1997) were defined as follows.

(iii) Target sequences for one-hybrid screening were previously described (Hayashi et al. 1999).

The following primer pairs were used for the polymerase chain reaction (PCR) to amplify the dPCNA promoter region after chromatin immunoprecipitation. A primer pair to amplify rp49 gene specific region was used for a control in PCNA amplification.

Plasmid construction

The plasmid, 5'–168DPCNAluc contains a dPCNA gene fragment spanning from –168 to +24 placed upstream of the luciferase (luc) gene in the firefly luciferase reporter plasmid, pGVB (Toyo Inc.). A set of 5'-deletion mutants of dPCNA promoter were constructed as follows: –149, –119, –116, and –70 DPCNAluc.

To construct the template for synthesizing double stranded RNA (dsRNA) for cut gene RNA interference, PCR products amplifying the first 750 nucleotides of the gene were inserted in KpnI and SmaI sites of pBluescript II SK(-). Two directions of cut gene fragment, named "forward" and "reverse," were used as templates after digestion with KpnI. RNAs were synthesized using a RiboMax kit (Promega) and annealed at 25 °C after incubating for 10 min at 70 °C. The construct and procedures to synthesize dsRNAs for the DREF gene were reported earlier (Kwon et al. 2003).

All plasmids were propagated in Escherichia coli (E. coli) XL-1 Blue, isolated by standard procedures (Sambrook et al. 1989) and further purified using a Qiagen Plasmid Midi Kit (Qiagen). The DNA sequencing was carried out with a BigDyeTM Terminator v3.0 Cycle sequencing Standard kit (Applied Biosystems) using an ABI PRISMTM 310 NT Genetic Analyzer (Applied Biosystems). When necessary, chemically synthesized oligonucleotides (17mer) were used as sequencing primers.

Antibodies

Expression of GST-Cut fusion protein in E. coli XL-1 Blue was carried out as described elsewhere (Smith & Johnson 1988). Lysates of cells were prepared and the GST-Cut fusion protein was purified by glutathione-Sepharose (Amersham Biotech) column chromatography as described (Smith & Johnson 1988). The purified GST-Cut fusion protein was used in mice to elicit production of polyclonal antibodies, which were purified from antiserum using E-Z-SEP (Amersham Biotech).

A monoclonal antibody against Drosophila Cut was purchased from Development Studies Hybridoma Bank, Iowa University. Monoclonal antibodies to DREF, Mab-1 and Mab-4 were raised as previously described (Hirose et al. 1993). Normal mouse IgG was purchased from Sigma.

Transient luciferase expression assays

Transient luciferase expression assays were carried out as described earlier (Yamaguchi et al. 1994; Hayashi et al. 1999) using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activities were normalized to Renilla luciferase activities. All transient expression data reported in this paper are means from three independent experiments, each performed in triplicate.

For dsRNA interference experiments, 1 x 105 S2 cells were plated in 24-well dishes in the presence of 3 µg/well of each dsRNA, DREF dsRNA, Cut dsRNA and Lac Z dsRNA in FBS free M3(BF) for 1 h. DsRNA free incubation (mock) was conducted as a control for 1 h in FBS free M3(BF). After the incubation, 4 volumes of M3(BF) medium containing 10% FBS were added to each well. Twenty-four hours after the RNAi treatment, the cells were co-transfected with reporter genes, 50 ng for each dPCNA-luc plasmid and 0.4 ng/well of plasmids containing the Renilla luciferase gene with the aid of Cell-Fectin reagents (Invitrogen). Cells were harvested 48 h after transfection and subjected to luciferase assay according to standard instructions with the kit. All assays were performed within the range of linear relation of activity to incubation time.

Chromatin Immunoprecipitation (ChIP)

ChIP assays were performed according to the manufacturer's manual (Upstate). Drosophila S2 cells were cultured for 24 h in M3(BF) (+10% FBS) media in the presence or absence of 10 µM of 20-hydroxyecdysone. 2 x 106 cells were collected and subjected to SDS-lysation after formaldehyde treatment for the cross linkage of DNA and proteins in 37 °C for 15 min. After sonication, 10 µg of antibodies or non-immune IgG were added and mixed in rotating tubes for 16 h. After washing with buffers of the ChIP assay kit (Low Salt Immune complex wash buffer, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl; High Salt Immune Complex Wash Buffer, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl; LiCl Immune Complex Wash Buffer, 0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1; TE Buffer, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Samples were then reverse crosslinked by 5 M NaCl treatment in 65 °C for 8 h. Purified template DNA samples were analyzed by PCR with specific primers for the dPCNA promoter or the rp49 gene as a negative control of PCR amplication.

Electrophoretic mobility shift assay

Preparation of Kc cell nuclear extracts and EMSA were performed as described earlier (Hirose et al. 1993) with minor modifications. A reaction mixture containing 20 mM HEPES pH 7.6, 100 mM KCl, 0.1 mM EDTA, 0.5 mM DTT, 10% glycerol, 0.5 µg of poly(dI-dC) was used with Kc cell nuclear extracts. 32P-labeled probes (10 000 cpm) were incubated in 15 µL of reaction mixture. To distinguish DREF-DNA and Cut-DNA complexes, Kc cell nuclear extracts were preincubated with anti-Cut polyclonal and anti-DREF monoclonal antibodies, respectively, for 1 h at 4 °C, and then reaction mixtures for EMSA were added. DNA-protein complexes were electrophoretically resolved on 4% polyacrylamide gels in 100 mM Tris-borate pH 8.3, 2 mM EDTA containing 2.5% glycerol at 25 °C. The gels were dried and then autoradiographed.

One-hybrid screening

The MATCHMAKER one-hybrid system protocol (Clontech) was used to prepare target reporter constructs, to integrate these constructs into Saccharomyces cerevisiae (S. cerevisiae) strain YM4271 (his– ura– leu–), and to screen an activation domain fusion library (pACT-cDNA library) from Drosophila third instar larvae (kindly supplied by Dr Elledge). Three copies of the double-stranded oligonucleotide DRE-P were placed in tandem upstream of the marker genes of both the pHISi-1 and pLacZi plasmids (Clontech). The two target reporter constructs were transformed into S. cerevisiae strain YM4271 in a consecutive manner to produce a dual reporter strain. This was transformed with the pACT-cDNA library and his+ ura+ leu+ transformants grown in synthetic dropout medium containing 40 mM 3-aminotriazole were selected. Each colony was streaked on synthetic dropout agar medium without histidine but containing 40 mM 3-aminotriazole and incubated for 3 days at 30 °C. A dry NEF-978X filter (NEN Life Science Products) was placed over the surface of each agar plate containing transformants. The filters were lifted off the agar plate and dipped in liquid nitrogen. The frozen colonies were then thawed at 25 °C and the filters were overlaid on to Whatman 5 filters that had been soaked in Z buffer (60 mM Na2HPO4·7H2O, 440 mM NaH2PO4·H2O, 10 mM KCl, 1 mM MgSO4·7H2O, 50 mMß-mercaptoethanol, pH 7.0) containing 0.03% 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) at 30 °C for 5–8 h. Positive blue colonies were selected. To confirm sequence specific interaction, plasmid DNA in each candidate was recovered in E. coli DH5 (Competent high, Toyobo) followed by retransformation into yeast strains containing the 5UREL reporter, the 3URE reporter, the 3DRE reporter, four copies of the CFDD-1 (4CFDD) reporter, or four copies of the E2F-P (4H2F) reporter (Hayashi et al. 1999).

Western immuno-blot analysis

Whole cell extracts from S2 cells prepared as described earlier (Kwon et al. 2000) were applied to SDS-polyacrylamide gels containing 7.5% acrylamide and transferred to polyvinylidene difluoride membranes (Bio-Rad) in transfer buffer (50 mM borate-NaOH, pH 9.0, 20% ethanol) at 4 °C for 16 h. The blotted membranes were incubated with culture supernatants of hybridomas producing monoclonal antibodies to DREF (Hirose et al. 1996) at 1 : 2000 dilution or Cut at 1 : 500 dilution, or monoclonal antibodies to rat PCNA (Sigma) at 1 : 1000 or tubulin IgG (Sigma) at 1 : 2000 for 1 h at 25 °C. The bound antibodies were detected with peroxidase-conjugated goat anti-mouse IgG and the ECL system (Amersham Pharmacia Biotech) according to the manufacturer's recommendations.

Double stranded RNA interference experiments were performed as described with some modifications (Worby et al. 2001; Kwon et al. 2003). 2 x 106 S2 cells were plated in 6 cm plates with 1 mL of serum free M3(BF) and incubated with 15 µg each of Cut, DREF or LacZ dsRNAs, or no dsRNA for 1 h at 25 °C. Five millilitre of 10% FBS M3(BF) was then added to each plate followed by incubation for 2 days, 3 days or 4 days at 25 °C in 5% CO2, before harvesting.


   Acknowledgements
 
We thank Drs H. Yoshida and F. Hirose for technical advice, Drs S.R. Fried and Y.N. Jan for supplying Cut cDNA, Dr S. Elledge for supplying pACT-Drosophila cDNA library, Dr M. Moore for comments on the English language in the manuscript, and all members in our laboratory for helpful discussion. This study was partially supported through Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government.


   Footnotes
 
Communicated by: Fumio Hanaoka

a Present address: Laboratory of Molecular Oncology, MGH Cancer Center Building 149, 13th Street, Charlestown, MA 02129- 2000, USA Back

* Correspondence: E-mail: myamaguc{at}kit.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Andres, V., Chiara, M.D. & Mahdavi, V. (1994) A new bipartite DNA-binding domain: cooperative interaction between the cut repeat and homeodomain of the cut homeo proteins. Genes Dev. 8, 245–257.[Abstract/Free Full Text]

Aufiero, B., Neufeld, E.J. & Orkin, S.H. (1994) Sequence-specific DNA binding of individual cut repeats of the human CCAAT displacement/cut homeodomain protein. Proc. Natl. Acad. Sci. USA 91, 7757–7761.[Abstract/Free Full Text]

Berger, E., Ringler, R., Alahiotis, S. & Frank, M. (1978) Ecdysone-induced changes in morphology and protein synthesis in Drosophila cell cultures. Dev. Biol. 62, 498–511.[CrossRef][Medline]

Blochlinger, K., Bodmer, R., Jack, J., Jan, L.Y. & Jan, Y.N. (1988) Primary structure and expression of a product from cut, a locus involved in specifying sensory organ identity in Drosophila. Nature 333, 629–635.[CrossRef][Medline]

Blochlinger, K., Jan, L.Y. & Jan, Y.N. (1991) Transformation of sensory organ identity by ectopic expression of Cut in Drosophila. Genes Dev. 5, 1124–1135.[Abstract/Free Full Text]

Blochlinger, K., Jan, L.Y. & Jan, Y.N. (1993) Postembryonic patterns of expression of cut, a locus regulating sensory organ identity in Drosophila. Development 117, 441–450.[Abstract]

Bodmer, R., Barbel, S., Sheperd, S., Jack, J.W., Jan, L.Y. & Jan, Y.N. (1987) Transformation of sensory organs by mutations of the cut locus of D. melanogaster. Cell 51, 293–307.[CrossRef][Medline]

Bray, S.J., Burke, B., Brown, N.H. & Hirsh, J. (1989) Embryonic expression pattern of a family of Drosophila proteins that interact with a central nervous system regulatory element. Genes Dev. 3, 1130–1145.[Abstract/Free Full Text]

Chattopadhyay, S., Whitehurst, C.E. & Chen, J. (1998) A nuclear matrix attachment region upstream of the T cell receptor beta gene enhancer binds Cux/CDP and SATB1 and modulates enhancer-dependent reporter gene expression but not endogenous gene expression. J. Biol. Chem. 273, 29838–29846.[Abstract/Free Full Text]

Cross, D.P. & Sang, J.H. (1978) Cell culture of individual Drosophila embryos. I. Development of wild type cultures. J. Embryol. Exp. Morph. 45, 161–172.[Medline]

Dickinson, L.A., Dickinson, C.D. & Kohwi-Shigematsu, T. (1997) An atypical homeodomain in SATB1 promotes specific recognition of the key structural element in a matrix attachment region. J. Biol. Chem. 272, 11463–11470.[Abstract/Free Full Text]

Durand, B., Vandaele, C., Spencer, D., Pantalacci, S. & Couble, P. (2000) Cloning and characterization of dRFX, the Drosophila member of the RFX family of transcription factors. Gene 246, 285–293.[CrossRef][Medline]

Dynlacht, B.D., Attardi, L.D., Admon, A., Freeman, M. & Tjian, R. (1989) Functional analysis of NTF-1, a developmentally regulated Drosophila transcription factor that binds neuronal cis elements. Genes Dev. 3, 1677–1688.[Abstract/Free Full Text]

Emery, P., Durand, B., Mach, B. & Reith, W. (1996) RFX proteins, a novel family of DNA binding proteins conserved in the eukaryotic kingdom. Nucleic Acids Res. 24, 803–807.[Abstract/Free Full Text]

Flores-Rozas, H., Kelman, Z., Dean, F., et al. (1994) Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase {delta} holoenzyme. Proc. Natl. Acad. Sci. USA 91, 8655–8659.[Abstract/Free Full Text]

Gvozdev, V.A., Gerasimova, T.I. & Birstein, V.J. (1974) Inactivation of 6-phosphogluconate dehydrogenase structural gene of Drosophila melanogaster caused by translocation to heterochromatin. Mol. Gen. Genet. 130, 251–260.[Medline]

Harada, R., Berube, G., Tamplin, O.J., Denis-Larose, C. & Nepveu, A. (1995) DNA-binding specificity of the cut repeats from the human cut-like protein. Mol. Cell. Biol. 15, 129–140.[Abstract]

Hart, C.M., Cuvier, O. & Laemmli, U.K. (1999) Evidence for an antagonistic relationship between the boundary element x associated factor BEAF and the transcription factor DREF. Chromosoma 108, 375–383.[CrossRef][Medline]

Hayashi, Y., Hirose, F., Nishimoto, Y., et al. (1997) Identification of CFDD (common regulatory factor for DNA replication and DREF genes) and role of its binding site in regulation of the proliferation cell nuclear antigen gene promoter. J. Biol. Chem. 272, 22848–22858.[Abstract/Free Full Text]

Hayashi, Y., Yamagishi, M., Nishimoto, Y., Taguchi, O., Matsukage, A. & Yamaguchi, M. (1999) A binding site for the transcription factor Grainyhead/Nuclear transcription factor-1 contributes to regulation of the Drosophila proliferating cell nuclear antigen gene promoter. J. Biol. Chem. 274, 35080–35088.[Abstract/Free Full Text]

Henderson, D.S., Banga, S.S., Grigliatti, T.A. & Boyd, J.B. (1994) Mutagen sensitivity and suppression of position-effect variegation result from mutations in mus209, the Drosophila gene encoding PCNA. EMBO J. 13, 1450–1459.[Medline]

Hirose, F., Yamaguchi, M., Handa, H., Inomata, Y. & Matsukage, A. (1993) Novel 8-base pair sequence (Drosophila DNA replication-related element) and specific binding factor involved in the expression of Drosophila genes for DNA polymerase {alpha} and proliferating cell nuclear antigen. J. Biol. Chem. 268, 2092–2099.[Abstract/Free Full Text]

Hirose, F., Yamaguchi, M., Kuroda, K., et al. (1996) Isolation and characterization of cDNA for DREF, a promoter-activating factor for Drosophila DNA replication-related genes. J. Biol. Chem. 271, 3930–3937.[Abstract/Free Full Text]

Hirose, F., Yamaguchi, M. & Matsukage, A. (1994) Repression of regulatory factor for Drosophila DNA replication-related gene promoter by zerknullt homeodomain protein. J. Biol. Chem. 269, 2937–2942.[Abstract/Free Full Text]

Huang, M., Zhou, Z. & Elledge, S.J. (1998) The DNA replication and damage checkpoint pathways induce transcription by inhibition of the Crt1 repressor. Cell 94, 595–605.[CrossRef][Medline]

Jaskulski, D., DeRiel, K., Mercer, W.E., Calabretta, B. & Baserga, R. (1988) Inhibition of cellular proliferation by antisense oligodeoxynucleotides to PCNA/cyclin. Science 240, 1544–1546.[Abstract/Free Full Text]

Kelman, Z. & Hurwitz, J. (1998) Protein–PCNA interactions: a DNA-scanning mechanism? Trends Biochem. Sci. 23, 236–238.[CrossRef][Medline]

Kelman, Z. & O'Donnell, M. (1995) Structural and functional similarities of prokaryotic and eukaryotic DNA polymerase sliding clamps. Nucleic Acids Res. 23, 3613–3620.[Abstract/Free Full Text]

Kwon, E., Hayashi, Y., Otsuki, K., et al. (2004) Armadillo/Pangolin regulates PCNA and DREF promoter activities. Biochim. Biophys. Acta 1679, 256–262.[Medline]

Kwon, E.J., Park, H.S., Kim, Y.S., et al. (2000) Transcriptional regulation of the Drosophila raf proto-oncogene by Drosophila STAT during development and in immune response. J. Biol. Chem. 275, 19824–19830.[Abstract/Free Full Text]

Kwon, E., Seto, H., Hirose, F., et al. (2003) Transcription control of a gene for Drosophila transcription factor, DREF by DRE and cis-elements conserved between Drosophila melanogaster and virilis. Gene 309, 101–116.[CrossRef][Medline]

Labrie, C., Lee, B.H. & Mathews, M.B. (1995) Transcription factor RFX1/EF-C and ATF1 associate with the adenovirus E1A-responsive element of the human proliferating cell nuclear antigen promoter. Nucleic Acids Res. 23, 3732–3741.[Abstract/Free Full Text]

Lee, B.H., Liu, M. & Mathews, M.B. (1998) Regulation of the human proliferating cell nuclear antigen promoter by the adenovirus E1A-associated protein p107. J. Virol. 72, 1138–1145.[Abstract/Free Full Text]

Liu, J., Bramblett, D., Zhu, Q., et al. (1997) The matrix attachment region-binding protein SATB1 participates in negative regulation of tissue-specific gene expression. Mol. Cell. Biol. 17, 5275–5287.[Abstract]

Liu, M., Lee, B.H. & Mathews, M.B. (1999) Involvement of RFX1 protein in the regulation of the human proliferating cell nuclear antigen promoter. J. Biol. Chem. 274, 15433–15439.[Abstract/Free Full Text]

Liu, Y.C., Marraccino, R.L., Keng, P.C., et al. (1989) Requirement for proliferating cell nuclear antigen expression during stages of the chinese hamster ovary cell cycle. Biochemistry 28, 2967–2974.[CrossRef][Medline]

Liu, S., McLeod, E. & Jack, J. (1991) Four distinct regulatory regions of the cut locus and their effect on cell type specification in Drosophila. Genetics 127, 151–159.[Abstract]

Ludlow, C., Choy, R. & Blochlinger, K. (1996) Functional analysis of Drosophila and mammalian cut proteins in files. Dev. Biol. 178, 149–159.[CrossRef][Medline]

Mailly, F., Berube, G., Harada, R., Mao, P.L., Phillips, S. & Nepveu, A. (1996) The human cut homeodomain protein can repress gene expression by two distinct mechanisms: active repression and competition for binding site occupancy. Mol. Cell. Biol. 16, 5346–5357.[Abstract]

Matsukage, A., Hirose, F., Hayashi, Y., Hamada, K. & Yamaguchi, M. (1995) The DRE sequence TATCGATA, a putative promoter-activating element for Drosophila melanogaster cell-proliferation-related genes. Gene 166, 233–236.[CrossRef][Medline]

Matsuoka, S., Yamaguchi, M. & Matsukage, A. (1994) D-type cyclin-binding regions of proliferating cell nuclear antigen. J. Biol. Chem. 269, 11030–11036.[Abstract/Free Full Text]

Moon, N.S., Berube, G. & Nepveu, A. (2000) CCAAT displacement activity involves CUT repeats 1 and 2, not the CUT homeodomain. J. Biol. Chem. 275, 31325–31334.[Abstract/Free Full Text]

Munsch, N. & Cade-Treyer, D. (1982) Effect of an ecdysteroid on DNA synthesis, DNA polymerase {alpha} and thymidine kinase activities of Drosophila melanogaster cells in vitro. Experientia 38, 1197–1199.[CrossRef][Medline]

Nepveu, A. (2001) Role of the multifunctional CDP/Cut/Cux homeodomain transcription factor in regulating differentiation, cell growth and development. Gene 270, 1–15.[CrossRef][Medline]

Neufeld, E.J., Skalnik, D.G., Lievens, P.M. & Orkin, S.H. (1992) Human CCAAT displacement protein is homologous to the Drosophila homeoprotein, cut. Nature Genet. 1, 50–55.[CrossRef][Medline]

Nishio, H. & Walsh, M.J. (2004) CCAAT displacement protein/cut homolog recruits G9a histone lysine methyltransferase to repress transcription. Proc. Natl. Acad. Sci. USA 101, 11257–11262.[Abstract/Free Full Text]

Ohno, K.H.F., Sakaguchi, K., Nishida, Y. & Matsukage, A. (1996) Transcriptional regulation of the Drosophila cycA gene by the DNA replication-related element (DRE) and DRE binding factor (DREF). Nucleic Acids Res. 24, 3942–3946.[Abstract/Free Full Text]

Otsuki, K., Hayashi, Y., Kato, M., Yoshida, H. & Yamaguchi, M. (2004) Characterization of dRFX2, a novel RFX family protein in Drosophila. Nucleic Acids Res. 32, 5636–5648.[Abstract/Free Full Text]

Reith, W., Herrero-Sanchez, C., Kobr, M., et al. (1990) MHC class II regulatory factor RFX has a novel DNA-binding domain and a functionally independent dimerization domain. Genes Dev. 4, 1528–1540.[Abstract/Free Full Text]

Rosset, R. (1978) Effects of ecdysone on a Drosophila cell line. Exp. Cell Res. 111, 31–36.[CrossRef][Medline]

Ryu, J.-R., Choi, T.-Y., Kwon, E.-J., et al. (1997) Transcriptional regulation of the Drosophila-raf protooncogene by the DNA replication-related element (DRE)/DRE-binding factor (DREF) system. Nucleic Acids Res. 24, 794–799.

Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

Shibahara, K. & Stillman, B. (1999) Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin. Cell 96, 575–585.[CrossRef][Medline]

Shivji, M.K.K., Kenny, M.K. & Wood, R.D. (1992) Proliferating cell nuclear antigen is required for DNA excision repair. Cell 69, 367–374.[CrossRef][Medline]

Smith, D.B. & Johnson, K.S. (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31–40.[CrossRef][Medline]

Takahashi, Y., Yamaguchi, M., Hirose, F., et al. (1996) DNA replication-related elements cooperate to enhance promoter activity of the Drosophila DNA polymerase {alpha} 73-kDa subunit gene. J. Biol. Chem. 271, 14541–14547.[Abstract/Free Full Text]

Takahashi, Y., Yamaguchi, M., Hirose, F., Kobayashi, J., Miyajima, S. & Matsukage, M. (1997) Involvement of the DNA replication-related element (DRE) and DRE-binding factor (DREF) in transcriptional regulation of the Bombyx mori PCNA gene. J. Biochem. 122, 1215–1223.[Abstract/Free Full Text]

Tsurimoto, T., Melendy, T. & Stillman, B. (1990) Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature 346, 534–539.[CrossRef][Medline]

Waga, S., Hannon, G.J., Beach, D. & Stillman, B. (1994) The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature 369, 574–578.[CrossRef][Medline]

Wang, Z., Goldstein, A., Zong, R.T., et al. (1999) Cux/CDP homeoprotein is a component of NF-muNR and represses the immunoglobulin heavy chain intronic enhancer by antagonizing the bright transcription activator. Mol. Cell. Biol. 19, 284–295.[Abstract/Free Full Text]

Warbrick, E. (1998) PCNA binding through a conserved motif. Bioessays 20, 195–199.[CrossRef][Medline]

Worby, C.A., Simonson-Leff, N. & Dixon, J.E. (2001) RNA interference of gene expression (RNAi) in cultured Drosophila cells. Sci. STKE 2001, PL1.

Wu, S.Y. & McLeod, M. (1995) The sak1+ gene of Schizosaccharomyces pombe encodes an RFX family DNA-binding protein that positively regulates cyclic AMP-dependent protein kinase mediated exit from the mitotic cycle. Mol. Cell. Biol. 15, 1479–1488.[Abstract]

Yamaguchi, M., Hayashi, Y., Matsuoka, S., Takahashi, T. & Matsukage, A. (1994) Differential effect of p53 on the promoters of mouse DNA polymerase ß gene and proliferating-cell-nuclear-antigen gene. Eur. J. Biochem. 221, 227–237.[Medline]

Yamaguchi, M., Hayashi, Y. & Matsukage, A. (1995a) Essential role of E2F recognition sites in regulation of the proliferating cell nuclear antigen gene promoter during Drosophila development. J. Biol. Chem. 270, 25159–25165.[Abstract/Free Full Text]

Yamaguchi, M., Hayashi, Y., Nishimoto, Y., Hirose, F. & Matsukage, A. (1995b) A nucleotide sequence essential for the function of DRE, a common promoter element for Drosophila DNA replication-related genes. J. Biol. Chem. 270, 15808–15814.[Abstract/Free Full Text]

Yamaguchi, M., Hirose, F. & Matsukage, A. (1996) Roles of multiple promoter elements of the proliferating cell nuclear antigen gene during Drosophila development. Genes Cells 1, 47–58.[Abstract]

Yamaguchi, M., Hirose, F., Nishida, Y. & Matsukage, A. (1991) Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknullt protein. Mol. Cell. Biol. 11, 4909–4917.[Abstract/Free Full Text]

Zhang, Z., Shibahara, K. & Stillman, B. (2000) PCNA connects DNA replication to epigenetic inheritance in yeast. Nature 408, 221–225.[CrossRef][Medline]




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